Title: Identification and Characterization of a Novel Nuclear Factor of Activated T-cells-1 Isoform Expressed in Mouse Brain
Abstract: The nuclear factor of activated T-cells (NFAT) family transcription factors play a key role in the control of cytokine gene expression in T-cells. Although initially identified in T-cells, recent data have unveiled unanticipated roles for NFATs in the development, proliferation, and differentiation of other tissues. Here we report the identification, cDNA cloning, and functional characterization of a new isoform of NFAT1 highly expressed in mouse brain. This isoform, which we named NFAT1-D, is identical to NFAT1 throughout the N-terminal regulatory domain and the portion of the Rel domain which includes the minimal region required for specific binding to DNA and interaction with AP-1. The homology stops sharply upstream of the 3′-boundary of the Rel homology domain and is followed by a short unique C-terminal region. NFAT1-D was expressed at high levels in all brain districts and was found as a constitutively active transcription complex. Transfection of a NFAT/luciferase reporter in the neuronal cell line PC12, which also expresses NFAT1-D, showed that these cells expressed a constitutive NFAT activity that was enhanced after nerve growth factor-induced differentiation but was resistant to the immunosuppressant cyclosporin A. NFAT1-D was, however, inducibly activated in a cyclosporin A-sensitive manner when expressed in T-cells, suggesting that the activity of NFAT proteins might be controlled by their specific cellular context. The nuclear factor of activated T-cells (NFAT) family transcription factors play a key role in the control of cytokine gene expression in T-cells. Although initially identified in T-cells, recent data have unveiled unanticipated roles for NFATs in the development, proliferation, and differentiation of other tissues. Here we report the identification, cDNA cloning, and functional characterization of a new isoform of NFAT1 highly expressed in mouse brain. This isoform, which we named NFAT1-D, is identical to NFAT1 throughout the N-terminal regulatory domain and the portion of the Rel domain which includes the minimal region required for specific binding to DNA and interaction with AP-1. The homology stops sharply upstream of the 3′-boundary of the Rel homology domain and is followed by a short unique C-terminal region. NFAT1-D was expressed at high levels in all brain districts and was found as a constitutively active transcription complex. Transfection of a NFAT/luciferase reporter in the neuronal cell line PC12, which also expresses NFAT1-D, showed that these cells expressed a constitutive NFAT activity that was enhanced after nerve growth factor-induced differentiation but was resistant to the immunosuppressant cyclosporin A. NFAT1-D was, however, inducibly activated in a cyclosporin A-sensitive manner when expressed in T-cells, suggesting that the activity of NFAT proteins might be controlled by their specific cellular context. Initially described as a transcriptional complex that bound a T-cell antigen receptor (TCR)1 response element on the interleukin (IL)-2 gene enhancer, nuclear factor of activated T-cells (NFAT) is a family of transcription factors crucially involved in the regulation of cytokine gene expression in T-cells (1Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2203) Google Scholar). NFAT activity is strongly unregulated after TCR triggering; however, receptor engagement can be bypassed by a combination of phorbol esters and calcium ionophores, which activate protein kinase C and induce a rise in intracellular calcium ions, respectively. This dual requirement reflects the subunit composition of NFAT factors, which includes a cytoplasmic and a nuclear component. In resting cells, NFAT is found as a cytosolic protein phosphorylated on serine residues. After an elevation in intracellular calcium ions, the calmodulin-dependent phosphatase calcineurin is activated and dephosphorylates NFAT, exposing a nuclear localization sequence near the N terminus of NFAT and resulting in its translocation to the nucleus (2Beals C.R. Clipstone N.A. Ho S.N. Crabtree G.R. Genes Dev. 1997; 11: 824-834Crossref PubMed Scopus (341) Google Scholar, 3Clipstone N.A. Crabtree G.R. Nature. 1992; 357: 695-697Crossref PubMed Scopus (1463) Google Scholar, 4Jain J. McCaffrey P.G. Miner Z. Kerppola T.K. Lambert J.N. Verdine G.L. Curran T. Rao A. Nature. 1993; 365: 352-355Crossref PubMed Scopus (673) Google Scholar, 5Loh C. Shaw K.T. Carew J. Viola J.P. Luo C. Perrino B.A. Rao A. J. Biol. Chem. 1996; 271: 10884-10891Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). This process is exquisitely sensitive to the immunosuppressants cylosporin A (CsA) and FK506, which interact with specific cytosolic receptors and form complexes that bind with high affinity to calcineurin and lock it in an inactive conformation (6Crabtree G.R. Cell. 1999; 96: 611-614Abstract Full Text Full Text PDF PubMed Scopus (661) Google Scholar, 7Flanagan W.M. Corthesy B. Bram R.J. Crabtree G.R. Nature. 1991; 352: 803-807Crossref PubMed Scopus (946) Google Scholar, 8Liu J. Farmer Jr., J.D. Lane W.S. Friedman J. Weissman I. Schreiber S.L. Cell. 1991; 66: 807-815Abstract Full Text PDF PubMed Scopus (3568) Google Scholar). In the nucleus NFAT assembles in cooperative DNA-binding complexes with dimers of the AP-1 family of transcription factors (4Jain J. McCaffrey P.G. Miner Z. Kerppola T.K. Lambert J.N. Verdine G.L. Curran T. Rao A. Nature. 1993; 365: 352-355Crossref PubMed Scopus (673) Google Scholar, 9Jain J. Valge-Archer V.E. Sinskey A.J. Rao A. J. Exp. Med. 1992; 175: 853-862Crossref PubMed Scopus (65) Google Scholar). Complementation studies using constitutively active mutants of signaling proteins have shown that the calcium/calcineurin and the Ras/protein kinase C/mitogen-activated protein kinase pathways are integrated at the level of NFAT proteins (10Baldari C.T. Heguy A. Telford J.L. J. Biol. Chem. 1993; 268: 2693-2698Abstract Full Text PDF PubMed Google Scholar, 11Woodrow M. Clipstone N.A. Cantrell D. J. Exp. Med. 1993; 178: 1517-1522Crossref PubMed Scopus (122) Google Scholar).The NFAT family of transcription factors includes to date five members, NFAT1 (also named NFATp or NFATc2), NFAT2 (also named NFATc or NFATc1), NFAT3 (also named NFATc4), NFAT4 (also named NFATc3), and the recently identified atypical member NFAT5 (12Ho S.N. Thomas D.J. Timmerman L.A. Li X. Francke U. Crabtree G.R. J. Biol. Chem. 1995; 270: 19898-19907Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 13Hoey T. Sun Y.L. Williamson K. Xu X. Immunity. 1995; 2: 461-472Abstract Full Text PDF PubMed Scopus (352) Google Scholar, 14Lopez-Rodriguez C. Aramburu J. Rakeman A.S. Rao A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7214-7219Crossref PubMed Scopus (312) Google Scholar, 15McCaffrey P.G. Luo C. Kerppola T.K. Jain J. Badalian T.M. Ho A.M. Burgeon E. Lane W.S. Lambert J.N. Curran T. Verdine G.L. Rao A. Hogen P.G. Science. 1993; 262: 750-754Crossref PubMed Scopus (376) Google Scholar, 16Masuda E.S. Naito Y. Tokumitsu H. Campbell D. Saito F. Hannum C. Arai K. Arai N. Mol. Cell. Biol. 1995; 15: 2697-2706Crossref PubMed Scopus (198) Google Scholar, 17Northrop J.P. Ho S.N. Chen L. Thomas D.J. Timmerman L.A. Nolan G.P. Admon A. Crabtree G.R. Nature. 1994; 369: 497-502Crossref PubMed Scopus (522) Google Scholar, 18Park J. Takeuchi A. Sharma S. J. Biol. Chem. 1996; 271: 20914-20921Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). All NFAT proteins share a conserved DNA binding domain, which shows a weak similarity with the DNA binding domain of Rel family proteins (RHD) and permits interaction with Fos/Jun heterodimers at composite DNA binding sites on a number of cytokine gene enhancers (1Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2203) Google Scholar). The N-terminal region shows significant homology among NFAT1–4 and is characterized by a number of features important for regulation, including a nuclear localization sequence, the sites of interaction with calcineurin, and a highly conserved SP repeat region (SPXXSPXXSPXXXXX(D/E)(D/E), which is likely to be the target of kinase/phosphatase activity (1Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2203) Google Scholar). Furthermore, the N-terminus of NFAT1 has been functionally characterized as a transactivation domain and, like the corresponding regions of NFAT2–4, contains at least one acidic/hydrophobic patch that resembles those implicated in transactivation by acidic activation domains (1Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2203) Google Scholar). In agreement with a regulatory role for the N-terminal NFAT homology region, NFAT5, which lacks this region, has a calcineurin-independent constitutive nuclear localization in a number of cell types, including T-cells (14Lopez-Rodriguez C. Aramburu J. Rakeman A.S. Rao A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7214-7219Crossref PubMed Scopus (312) Google Scholar). NFAT family members differ widely at their C termini, as they are expressed mostly as multiple isoforms generated by either alternative splicing or, as recently shown for NFAT2, by alternative polyadenylation (19Chuvpilo S. Zimmer M. Kerstan A. Glockner J. Avots A. Escher C. Fischer C. Inashkina I. Jankevics E. Berberich-Siebelt F. Schmitt E. Serfling E. Immunity. 1999; 10: 261-269Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). The difference in transactivation activity among NFAT isoforms appears related to the presence, in specific isoforms, of an additional transactivation domain in the C-terminal region (20Luo C. Burgeon E. Rao A. J. Exp. Med. 1996; 184: 141-147Crossref PubMed Scopus (86) Google Scholar).Although initially characterized in T-cells, NFAT family members are in some instances broadly expressed not only in other cells of the immune system, but also in ontogenically distinct tissues. While NFAT2 expression is mostly restricted to T and B-cells and NFAT4 to thymocytes (12Ho S.N. Thomas D.J. Timmerman L.A. Li X. Francke U. Crabtree G.R. J. Biol. Chem. 1995; 270: 19898-19907Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 18Park J. Takeuchi A. Sharma S. J. Biol. Chem. 1996; 271: 20914-20921Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), NFAT1, NFAT3, and NFAT5 are found in many tissues in both man and mouse (12Ho S.N. Thomas D.J. Timmerman L.A. Li X. Francke U. Crabtree G.R. J. Biol. Chem. 1995; 270: 19898-19907Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 13Hoey T. Sun Y.L. Williamson K. Xu X. Immunity. 1995; 2: 461-472Abstract Full Text PDF PubMed Scopus (352) Google Scholar, 14Lopez-Rodriguez C. Aramburu J. Rakeman A.S. Rao A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7214-7219Crossref PubMed Scopus (312) Google Scholar, 21Boss V. Abbott K.L. Wang X.F. Pavlath G.K. Murphy T.J. J. Biol. Chem. 1998; 273: 19664-19671Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 22Ho A.M. Jain J. Rao A. Hogan P.G. J. Biol. Chem. 1994; 269: 28181-28186Abstract Full Text PDF PubMed Google Scholar), and unanticipated roles for these factors have been unveiled in development, proliferation, and differentiation of a number of tissues. For example, NFAT1 has been shown to be a repressor of chondrogenesis (23Ranger A.M. Gerstenfeld L.C. Wang J. Kon T. Bae H. Gravallese E.M. Glimcher M.J. Glimcher L.H. J. Exp. Med. 2000; 191: 9-22Crossref PubMed Scopus (163) Google Scholar), whereas NFAT2 was found to be essential for heart development (24de la Pompa J.L. Timmerman L.A. Takimoto H. Yoshida H. Elia A.J. Samper E. Potter J. Wakeham A. Marengere L. Langille B.L. Crabtree G.R. Mak T.W. Nature. 1998; 392: 182-186Crossref PubMed Scopus (542) Google Scholar, 25Ranger A.M. Grusby M.J. Hodge M.R. Gravallese E.M. de la Brousse F.C. Hoey T. Mickanin C. Baldwin H.S. Glimcher L.H. Nature. 1998; 392: 186-190Crossref PubMed Scopus (508) Google Scholar). Furthermore, a number of data suggest a potential role for NFAT factors in myogenesis and adipogenesis (26Abbott K.L. Friday B.B. Thaloor D. Murphy T.J. Pavlath G.K. Mol. Biol. Cell. 1998; 9: 2905-2916Crossref PubMed Scopus (196) Google Scholar, 27Ho I.-C. Kim H.H.-J. Spiegelman B.M. Glimcher L.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15537-15541Crossref PubMed Scopus (97) Google Scholar). Specific NFAT family members are also expressed in the brain. NFAT1 has been detected in the mouse olfactory bulb and in a neuronal cell line (22Ho A.M. Jain J. Rao A. Hogan P.G. J. Biol. Chem. 1994; 269: 28181-28186Abstract Full Text PDF PubMed Google Scholar), and NFAT3- and NFAT5-specific mRNA has also been detected at high levels in the brain (13Hoey T. Sun Y.L. Williamson K. Xu X. Immunity. 1995; 2: 461-472Abstract Full Text PDF PubMed Scopus (352) Google Scholar, 14Lopez-Rodriguez C. Aramburu J. Rakeman A.S. Rao A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7214-7219Crossref PubMed Scopus (312) Google Scholar). Recently NFAT3 has been shown to translocate to the nucleus and activate NFAT-dependent transcription in response to electrical activity or potassium depolarization in pyramidal neurons, suggesting a role for NFAT3 in hippocampal synaptic plasticity and memory (28Graef I.A. Mermelstein P.G. Stankunas K. Neilson J.R. Deisseroth K. Tsien R.W. Crabtree G.R. Nature. 1999; 401: 703-708Crossref PubMed Scopus (451) Google Scholar). Hence, NFAT factors might be implicated in brain development and function. Here we report the cDNA cloning and characterization of a new NFAT1 isoform highly expressed and constitutively active in mouse brain, initially suggested by the presence of high levels of constitutive luciferase activity in the brain of a NFAT/luciferase (NFAT/luc) reporter transgenic mouse. Initially described as a transcriptional complex that bound a T-cell antigen receptor (TCR)1 response element on the interleukin (IL)-2 gene enhancer, nuclear factor of activated T-cells (NFAT) is a family of transcription factors crucially involved in the regulation of cytokine gene expression in T-cells (1Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2203) Google Scholar). NFAT activity is strongly unregulated after TCR triggering; however, receptor engagement can be bypassed by a combination of phorbol esters and calcium ionophores, which activate protein kinase C and induce a rise in intracellular calcium ions, respectively. This dual requirement reflects the subunit composition of NFAT factors, which includes a cytoplasmic and a nuclear component. In resting cells, NFAT is found as a cytosolic protein phosphorylated on serine residues. After an elevation in intracellular calcium ions, the calmodulin-dependent phosphatase calcineurin is activated and dephosphorylates NFAT, exposing a nuclear localization sequence near the N terminus of NFAT and resulting in its translocation to the nucleus (2Beals C.R. Clipstone N.A. Ho S.N. Crabtree G.R. Genes Dev. 1997; 11: 824-834Crossref PubMed Scopus (341) Google Scholar, 3Clipstone N.A. Crabtree G.R. Nature. 1992; 357: 695-697Crossref PubMed Scopus (1463) Google Scholar, 4Jain J. McCaffrey P.G. Miner Z. Kerppola T.K. Lambert J.N. Verdine G.L. Curran T. Rao A. Nature. 1993; 365: 352-355Crossref PubMed Scopus (673) Google Scholar, 5Loh C. Shaw K.T. Carew J. Viola J.P. Luo C. Perrino B.A. Rao A. J. Biol. Chem. 1996; 271: 10884-10891Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). This process is exquisitely sensitive to the immunosuppressants cylosporin A (CsA) and FK506, which interact with specific cytosolic receptors and form complexes that bind with high affinity to calcineurin and lock it in an inactive conformation (6Crabtree G.R. Cell. 1999; 96: 611-614Abstract Full Text Full Text PDF PubMed Scopus (661) Google Scholar, 7Flanagan W.M. Corthesy B. Bram R.J. Crabtree G.R. Nature. 1991; 352: 803-807Crossref PubMed Scopus (946) Google Scholar, 8Liu J. Farmer Jr., J.D. Lane W.S. Friedman J. Weissman I. Schreiber S.L. Cell. 1991; 66: 807-815Abstract Full Text PDF PubMed Scopus (3568) Google Scholar). In the nucleus NFAT assembles in cooperative DNA-binding complexes with dimers of the AP-1 family of transcription factors (4Jain J. McCaffrey P.G. Miner Z. Kerppola T.K. Lambert J.N. Verdine G.L. Curran T. Rao A. Nature. 1993; 365: 352-355Crossref PubMed Scopus (673) Google Scholar, 9Jain J. Valge-Archer V.E. Sinskey A.J. Rao A. J. Exp. Med. 1992; 175: 853-862Crossref PubMed Scopus (65) Google Scholar). Complementation studies using constitutively active mutants of signaling proteins have shown that the calcium/calcineurin and the Ras/protein kinase C/mitogen-activated protein kinase pathways are integrated at the level of NFAT proteins (10Baldari C.T. Heguy A. Telford J.L. J. Biol. Chem. 1993; 268: 2693-2698Abstract Full Text PDF PubMed Google Scholar, 11Woodrow M. Clipstone N.A. Cantrell D. J. Exp. Med. 1993; 178: 1517-1522Crossref PubMed Scopus (122) Google Scholar). The NFAT family of transcription factors includes to date five members, NFAT1 (also named NFATp or NFATc2), NFAT2 (also named NFATc or NFATc1), NFAT3 (also named NFATc4), NFAT4 (also named NFATc3), and the recently identified atypical member NFAT5 (12Ho S.N. Thomas D.J. Timmerman L.A. Li X. Francke U. Crabtree G.R. J. Biol. Chem. 1995; 270: 19898-19907Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 13Hoey T. Sun Y.L. Williamson K. Xu X. Immunity. 1995; 2: 461-472Abstract Full Text PDF PubMed Scopus (352) Google Scholar, 14Lopez-Rodriguez C. Aramburu J. Rakeman A.S. Rao A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7214-7219Crossref PubMed Scopus (312) Google Scholar, 15McCaffrey P.G. Luo C. Kerppola T.K. Jain J. Badalian T.M. Ho A.M. Burgeon E. Lane W.S. Lambert J.N. Curran T. Verdine G.L. Rao A. Hogen P.G. Science. 1993; 262: 750-754Crossref PubMed Scopus (376) Google Scholar, 16Masuda E.S. Naito Y. Tokumitsu H. Campbell D. Saito F. Hannum C. Arai K. Arai N. Mol. Cell. Biol. 1995; 15: 2697-2706Crossref PubMed Scopus (198) Google Scholar, 17Northrop J.P. Ho S.N. Chen L. Thomas D.J. Timmerman L.A. Nolan G.P. Admon A. Crabtree G.R. Nature. 1994; 369: 497-502Crossref PubMed Scopus (522) Google Scholar, 18Park J. Takeuchi A. Sharma S. J. Biol. Chem. 1996; 271: 20914-20921Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). All NFAT proteins share a conserved DNA binding domain, which shows a weak similarity with the DNA binding domain of Rel family proteins (RHD) and permits interaction with Fos/Jun heterodimers at composite DNA binding sites on a number of cytokine gene enhancers (1Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2203) Google Scholar). The N-terminal region shows significant homology among NFAT1–4 and is characterized by a number of features important for regulation, including a nuclear localization sequence, the sites of interaction with calcineurin, and a highly conserved SP repeat region (SPXXSPXXSPXXXXX(D/E)(D/E), which is likely to be the target of kinase/phosphatase activity (1Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2203) Google Scholar). Furthermore, the N-terminus of NFAT1 has been functionally characterized as a transactivation domain and, like the corresponding regions of NFAT2–4, contains at least one acidic/hydrophobic patch that resembles those implicated in transactivation by acidic activation domains (1Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2203) Google Scholar). In agreement with a regulatory role for the N-terminal NFAT homology region, NFAT5, which lacks this region, has a calcineurin-independent constitutive nuclear localization in a number of cell types, including T-cells (14Lopez-Rodriguez C. Aramburu J. Rakeman A.S. Rao A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7214-7219Crossref PubMed Scopus (312) Google Scholar). NFAT family members differ widely at their C termini, as they are expressed mostly as multiple isoforms generated by either alternative splicing or, as recently shown for NFAT2, by alternative polyadenylation (19Chuvpilo S. Zimmer M. Kerstan A. Glockner J. Avots A. Escher C. Fischer C. Inashkina I. Jankevics E. Berberich-Siebelt F. Schmitt E. Serfling E. Immunity. 1999; 10: 261-269Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). The difference in transactivation activity among NFAT isoforms appears related to the presence, in specific isoforms, of an additional transactivation domain in the C-terminal region (20Luo C. Burgeon E. Rao A. J. Exp. Med. 1996; 184: 141-147Crossref PubMed Scopus (86) Google Scholar). Although initially characterized in T-cells, NFAT family members are in some instances broadly expressed not only in other cells of the immune system, but also in ontogenically distinct tissues. While NFAT2 expression is mostly restricted to T and B-cells and NFAT4 to thymocytes (12Ho S.N. Thomas D.J. Timmerman L.A. Li X. Francke U. Crabtree G.R. J. Biol. Chem. 1995; 270: 19898-19907Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 18Park J. Takeuchi A. Sharma S. J. Biol. Chem. 1996; 271: 20914-20921Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), NFAT1, NFAT3, and NFAT5 are found in many tissues in both man and mouse (12Ho S.N. Thomas D.J. Timmerman L.A. Li X. Francke U. Crabtree G.R. J. Biol. Chem. 1995; 270: 19898-19907Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 13Hoey T. Sun Y.L. Williamson K. Xu X. Immunity. 1995; 2: 461-472Abstract Full Text PDF PubMed Scopus (352) Google Scholar, 14Lopez-Rodriguez C. Aramburu J. Rakeman A.S. Rao A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7214-7219Crossref PubMed Scopus (312) Google Scholar, 21Boss V. Abbott K.L. Wang X.F. Pavlath G.K. Murphy T.J. J. Biol. Chem. 1998; 273: 19664-19671Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 22Ho A.M. Jain J. Rao A. Hogan P.G. J. Biol. Chem. 1994; 269: 28181-28186Abstract Full Text PDF PubMed Google Scholar), and unanticipated roles for these factors have been unveiled in development, proliferation, and differentiation of a number of tissues. For example, NFAT1 has been shown to be a repressor of chondrogenesis (23Ranger A.M. Gerstenfeld L.C. Wang J. Kon T. Bae H. Gravallese E.M. Glimcher M.J. Glimcher L.H. J. Exp. Med. 2000; 191: 9-22Crossref PubMed Scopus (163) Google Scholar), whereas NFAT2 was found to be essential for heart development (24de la Pompa J.L. Timmerman L.A. Takimoto H. Yoshida H. Elia A.J. Samper E. Potter J. Wakeham A. Marengere L. Langille B.L. Crabtree G.R. Mak T.W. Nature. 1998; 392: 182-186Crossref PubMed Scopus (542) Google Scholar, 25Ranger A.M. Grusby M.J. Hodge M.R. Gravallese E.M. de la Brousse F.C. Hoey T. Mickanin C. Baldwin H.S. Glimcher L.H. Nature. 1998; 392: 186-190Crossref PubMed Scopus (508) Google Scholar). Furthermore, a number of data suggest a potential role for NFAT factors in myogenesis and adipogenesis (26Abbott K.L. Friday B.B. Thaloor D. Murphy T.J. Pavlath G.K. Mol. Biol. Cell. 1998; 9: 2905-2916Crossref PubMed Scopus (196) Google Scholar, 27Ho I.-C. Kim H.H.-J. Spiegelman B.M. Glimcher L.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15537-15541Crossref PubMed Scopus (97) Google Scholar). Specific NFAT family members are also expressed in the brain. NFAT1 has been detected in the mouse olfactory bulb and in a neuronal cell line (22Ho A.M. Jain J. Rao A. Hogan P.G. J. Biol. Chem. 1994; 269: 28181-28186Abstract Full Text PDF PubMed Google Scholar), and NFAT3- and NFAT5-specific mRNA has also been detected at high levels in the brain (13Hoey T. Sun Y.L. Williamson K. Xu X. Immunity. 1995; 2: 461-472Abstract Full Text PDF PubMed Scopus (352) Google Scholar, 14Lopez-Rodriguez C. Aramburu J. Rakeman A.S. Rao A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7214-7219Crossref PubMed Scopus (312) Google Scholar). Recently NFAT3 has been shown to translocate to the nucleus and activate NFAT-dependent transcription in response to electrical activity or potassium depolarization in pyramidal neurons, suggesting a role for NFAT3 in hippocampal synaptic plasticity and memory (28Graef I.A. Mermelstein P.G. Stankunas K. Neilson J.R. Deisseroth K. Tsien R.W. Crabtree G.R. Nature. 1999; 401: 703-708Crossref PubMed Scopus (451) Google Scholar). Hence, NFAT factors might be implicated in brain development and function. Here we report the cDNA cloning and characterization of a new NFAT1 isoform highly expressed and constitutively active in mouse brain, initially suggested by the presence of high levels of constitutive luciferase activity in the brain of a NFAT/luciferase (NFAT/luc) reporter transgenic mouse. We acknowledge Sonia Grassini for technical assistance, Cristina Ulivieri for assistance and advice on the confocal microscopy, Silvia Guidotti for automatic sequencing, Aldo Muzzi for oligonucleotide synthesis, and Giancarlo Benocci for secretarial assistance. We also thank Ed Palmer and Jerry Crabtree for the antibodies, Stefano Alemà for nerve growth factor and for useful suggestions, and Edgar Serfling and Andris Avots for reagents and productive discussions. T-cell antigen receptor interleukin nuclear factor of activated T-cells cyclosporin A luciferase glutathioneS-transferase reverse transcription polymerase chain reaction monoclonal antibody phorbol 12-myristate 13-acetate green fluorescent protein